Alimentary protein-based scaffolds (APS) for wound healing, regenerative medicine and drug discovery

ABSTRACT

The invention provides engineered biomaterials derived from plant products. The engineered biomaterials are useful for biomedical applications. The engineered biomaterials are able to support the growth of animal cells.

CROSS-REFERENCE TO RELATED APPLICATIONS

This is a National Stage application of PCT International ApplicationNo. PCT/US2008/001936, filed Feb. 14, 2008, which in turn claims thebenefit pursuant to 35 U.S.C. §119(e) of U.S. Provisional ApplicationNo. 60/889,782, filed on Feb. 14, 2007 which is hereby incorporated byreference in its entirety herein.

BACKGROUND OF THE INVENTION

Tissue engineering aims at restoring, maintaining or improving tissuefunction so as to extend and/or preserve the well being of an individualwhile decreasing the major cost burden on the medical community. Thesenatural processes are occurring in nature using the 3D-structure ofextracellular matrix (ECM) (the natural scaffold), which allows cells togrow, proliferate and differentiate within it. Artificial scaffolds havebeen made and used for therapeutic purposes (i.e. cardiac or skinimplants) from natural polymers that desorb or degrade within the body.

The major challenge for tissue engineering researchers is to findmaterials and processing techniques that allow them to produce ECMmimicking scaffolds that promote cell growth and organization into aspecific architecture, inducing differentiated cell function. ECM is acomplex three-dimensional ultrastructure of proteins, proteoglycans andglycoproteins, used for cells growth in native tissue. In fact, thereare many different types of ECMs for different parts of the body, forexample, fibrous proteins are dominant material in tendon,polysaccharides are found largely existing in cartilage and so theforth. Collagens have been found to be the key proteins in ECM and alsoare the most ample proteins in the whole body.

ECM provides attachment sites and mechanical support for cells. Thetopology of ECM has been found to affect the cell structure,functionality and its physiological responsiveness. The geometry of thenatural matrix was reported to modulate the cell polarity. Thyroidcells, smooth muscle cell and hepatocytes are different types of cellsfound to be affected by ECM's topology, with 3D-structures inducing celldifferentiation more effectively than 2D configurations. The arrangementof ECM's configuration involves multiple length scales, layers andmorphologies. However, although much is know about 3-D scaffolding ofmaterials according to ECM topology to proliferate cell growth,satisfactory techniques and/or synthetic scaffolds have not been easilyto construct.

In human skin, dermal fibroblasts secrete keratinocyte growth factor(KGF) and other growth factors that regulate keratinocyte proliferationand migration (Huang, et al., 2005, J Biomed Sci. 12(6): 855-67), whilekeratinocyte-derived cytokines may downregulate collagen synthesis byfibroblasts (Harrison, et al., 2006, Br J Dermatol. 154(3): 401-10). Inlarge or non-healing wounds, this epithelial-mesenchymal interaction isobstructed by the lack of physical and biochemical cues for host cellsto migrate and repair the wound site. Hence, an implantable platform isneeded to provide an environment inductive to skin regeneration, byrecruiting host cells and inducing them to secrete the appropriatesignals and matrix components for repair.

Accordingly, there is a need for bioengineered tissue substitutes thatcan be custom-engineered to match the biomechanical, biochemical, andbiological needs of the specific tissue or organ they are designed toreplace.

BRIEF SUMMARY OF THE INVENTION

The present invention provides an electroprocessed compositioncomprising fibers of plant product derived biomaterials. In oneembodiment, the invention provides an electrospun composition comprisingfibers of plant product derived biomaterials.

In one embodiment, the plant product is from a plant selected from thegroup consisting of corn, wheat, potato, sorghums, tapioca, rice, arrowroot, sago, soybean, pea, sunflower, peanut, gelatin, and anycombination thereof.

In one embodiment, the plant product is soy protein isolate. In anotherembodiment, the plant product is corn zein.

In one embodiment, the composition is capable of supporting cell growth.In another embodiment, the composition is capable of supporting themaintenance of a differentiation state of a cell.

In one embodiment, the composition further comprises a cell. In anotherembodiment, the cell is genetically modified.

In one embodiment, the composition comprises a material selected fromthe group consisting of fibronectin, laminin, collagen, glycoprotein,thrombospondin, elastin, fibrillin, mucopolysaccharide, glycolipid,heparin sulfate, chondroitin sulfate, keratin sulfate,glycosaminoglycan, hyaluronic acid, proteoglycan, vitronectin,poly-D-lysine, polysaccharide, and any combination thereof.

In one embodiment, the composition comprises a synthetic material. Inanother embodiment, the synthetic material is selected from the groupconsisting of poly(epsilon-caprolactone) (PCL), poly(lactic acid) (PLA),poly(glycolic acid) (PGA), copolymers poly(lactide-co-glycolide) (PLGA),polyaniline, poly(ethylene oxide) (PEO), and any combination thereof.

The invention also provides a method of making a composition comprisinga biomaterial derived from a plant product, wherein the biomaterial iselectroprocessed to produce electroprocessed fibers. The methodcomprises obtaining a plant product and dissolving the plant product ina solvent to produce a protein solution; and subjecting the proteinsolution to electroprocessing to produced electroprocessed fibers.

In one embodiment, the step of electroprocessing is electrospinning; andthe electroprocessed fibers are electrospun fibers.

In one embodiment, the plant product is from a plant selected from thegroup consisting of corn, wheat, potato, sorghums, tapioca, rice, arrowroot, sago, soybean, pea, sunflower, peanut, gelatin, and anycombination thereof.

In one embodiment, the plant product is soy protein isolate. In anotherembodiment, the soy protein isolate is blended with poly(ethylene oxide)(PEO).

In one embodiment, the plant product is corn zein.

In one embodiment, the biomaterial comprises a material selected fromthe group consisting of fibronectin, laminin, collagen, glycoprotein,thrombospondin, elastin, fibrillin, mucopolysaccharide, glycolipid,heparin sulfate, chondroitin sulfate, keratin sulfate,glycosaminoglycan, hyaluronic acid, proteoglycan, vitronectin,poly-D-lysine, polysaccharide, and any combination thereof.

In one embodiment, the biomaterial further comprises a syntheticmaterial. In another embodiment, the synthetic material is selected fromthe group consisting of poly(epsilon-caprolactone) (PCL), poly(lacticacid) (PLA), poly(glycolic acid) (PGA), copolymerspoly(lactide-co-glycolide) (PLGA), polyaniline, poly(ethylene oxide)(PEO), and any combination thereof.

In one embodiment, the solvent is selected from the group consisting ofan organic solvent, an acid, a base, an alcohol, and any combinationthereof. In another embodiment, the solvent is selected from the groupconsisting of 1,1,1,3,3,3-hexafluoro-2-propanol (HFP) and glacial aceticacid.

The invention also provides a method of culturing a cell with anengineered scaffold comprising a biomaterial derived from a plantproduct, wherein the biomaterial is electroprocssed to produceelectroprocessed fibers. The method comprises contacting cells with theengineered scaffold in the presence of a culture medium.

In one embodiment, the culturing of a cell with a scaffold produces atarget tissue substitute. In another embodiment, the cell is selectedfrom the group consisting of stem cells, muscle cells, endothelialcells, nerve cells, bone cells, heart cells, epithelial cells,fibroblasts, and mixtures thereof.

The invention also provides a method of delivering an agent to a mammal.The method comprises administering an engineered scaffold comprising abiomaterial derived from a plant product, wherein the biomaterial iselectroprocessed to produce electroprocessed fibers, further wherein thescaffold comprises an agent.

In one embodiment, the agent is a cell. In another embodiment, the agentis selected from the group consisting of an extracellular matrixcomponent, a growth factor, a differentiation factor, and combinationsthereof. In another embodiment, the agent is selected from the groupconsisting of a chemical agent, a pharmaceutical, a peptide, a nucleicacid, and any combination thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

For the purpose of illustrating the invention, there are depicted in thedrawings certain embodiments of the invention. However, the invention isnot limited to the precise arrangements and instrumentalities of theembodiments depicted in the drawings.

FIG. 1, comprising FIGS. 1A-1H, is a series of images demonstrating thegeneral trend of increase in fiber width or diameter corresponds withincreasing protein concentration. FIG. 1A is an image of fibermorphology of electrospun fibers of 5% SPI, 0.025% PEO blended fiber;FIG. 1B is an image of fiber morphology electrospun fibers of 6% SPI,0.025% PEO blended fiber; FIG. 1C is an image of fiber morphology ofelectrospun fibers of 7% SPI, 0.025% blended fiber; FIG. 1D is an imageof fiber morphology of electrospun fibers of 8% SPI, 0.025% blendedfiber; FIG. 1E is an image of fiber morphology of electrospun fibers of5% SPI, 0.05% PEO blended fiber; FIG. 1F is an image of fiber morphologyof electrospun fibers of 6% SPI, 0.05% PEO blended fiber; FIG. 1G is animage of fiber morphology of electrospun fibers of 7% SPI, 0.05% PEOblended fiber; and FIG. 1H is an image of fiber morphology ofelectrospun fibers of 8% SPI, 0.05% PEO blended fiber.

FIG. 2 is a chart demonstrating the general trend of increase in fiberwidth or diameter corresponds with increasing protein concentration ofsoy protein isolate (SPI) when blended with either 0.0025% PEO or 0.05%PEO.

FIG. 3 is a chart demonstrating the general trend of increase in fiberwidth or diameter corresponds with increasing protein concentration ofzein.

FIG. 4 is a chart depicting the variation of fiber diameter followinghydration in DMEM, PBS, and water for 2 hours and 24 hours.

FIG. 5, comprising FIGS. 5A-5F, is a series of images demonstrating thatthe engineered scaffolds can support cell growth. FIG. 5A is an imagedepicting the growth of human dermal fibroblasts on electrospun fibersof 5% SPI, 0.05% PEO blended fiber; FIG. 5B is an image depicting thegrowth of human dermal fibroblasts on electrospun fibers of 6% SPI,0.05% PEO blended fiber; FIG. 5C is an image depicting the growth ofhuman dermal fibroblasts on electrospun fibers 7% SPI, 0.05% PEO blendedfiber; FIG. 5D is an image depicting the growth of human dermalfibroblasts on electrospun fibers 8% SPI, 0.05% PEO blended fiber; FIG.5E is an image depicting the growth of human dermal fibroblasts onelectrospun fibers of 8% gelatin; FIG. 5F is an image depicting thegrowth of human dermal fibroblasts on electrospun fibers of 20% PLGA.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is partly based on the discovery thatplant-derived proteins can be used as a source of biomaterials fortissue engineering purposes. In one aspect, the invention includes ascaffold produced from a plant product, wherein the scaffold is able tosupport the growth of animal cells. In another aspect, the scaffoldmimics natural extracellular matrix (ECM).

The invention includes the use of any products obtained from alimentaryplants. Non-limiting examples of alimentary plants include, but are notlimited to corn, wheat, potato, sorghums, tapioca, rice, arrow root,sago, soybean, pea, sunflower, peanut, gelatin.

In one embodiment of the invention, the scaffold is produced by anelectrospinning process. In certain aspects, the electrospinning processof the present invention uses a one-step electrospinning technique andtherefore is easy to use and is cost effective.

The invention also provides fibers and nanofibrous biocompatible matrixelectrospun from a blend of synthetic polymers and natural proteins. Thematrix can be used as tissue engineering scaffold and implanted into thebody to replace/repair damaged/non-functional tissues. The particularblends provide a unique mix of mechanical and physical properties thatfacilitates cell penetration and proliferation within the scaffoldswithout crosslinking.

Electrospinning provides an efficient approach to fabricating scaffoldsderived from proteins of plants for tissue engineering. The advantage ofthis scaffold is the accessibility of proteinaceous products fromplants. The novel approach to generating composite scaffolds ofplant-based biomaterials affords tissue engineers the ability to meetall necessary design criteria in fabricating scaffolds for a givenapplication, such as drug delivery, wound healing, regenerativemedicine, and the like.

DEFINITIONS

Unless defined otherwise, all technical and scientific terms used hereingenerally have the same meaning as commonly understood by one ofordinary skill in the art to which this invention belongs. Generally,the nomenclature used herein and the laboratory procedures in cellculture, molecular genetics, organic chemistry, and nucleic acidchemistry and hybridization are those well known and commonly employedin the art.

Standard techniques are used for nucleic acid and peptide synthesis. Thetechniques and procedures are generally performed according toconventional methods in the art and various general references (e.g.,Sambrook and Russell, 2001, Molecular Cloning, A Laboratory Approach,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., andAusubel et al., 2002, Current Protocols in Molecular Biology, John Wiley& Sons, New York, N.Y.), which are provided throughout this document.

The articles “a” and “an” are used herein to refer to one or to morethan one (i.e., to at least one) of the grammatical object of thearticle. By way of example, “an element” means one element or more thanone element.

The term “about” will be understood by persons of ordinary skill in theart and will vary to some extent based on the context in which it isused.

As used herein, “administering” refers to at least oral administration,administration as a suppository, topical contact, intravenous,intraperitoneal, intramuscular, intralesional, intranasal orsubcutaneous administration, or the implantation of a slow-releasedevice e.g., a mini-osmotic pump, to the subject.

The term “attached,” as used herein encompasses interaction including,but not limited to, covalent bonding, ionic bonding, chemisorption,physisorption and combinations thereof.

The term “biomolecule” or “bioorganic molecule” refers to an organicmolecule typically made by living organisms. This includes, for example,molecules comprising nucleotides, amino acids, sugars, fatty acids,steroids, nucleic acids, polypeptides, peptides, peptide fragments,carbohydrates, lipids, and combinations of these (e.g., glycoproteins,ribonucleoproteins, lipoproteins, or the like).

The term “differentiation factor”, as used herein, refers to a moleculethat induces a stem cell or progenitor cell to commit to a particularspecialized cell type.

“Extracellular matrix” or “matrix” refers to one or more substances thatprovide substantially the same conditions for supporting cell growth asprovided by an extracellular matrix synthesized by feeder cells. Thematrix may be provided on a substrate. Alternatively, the component(s)comprising the matrix may be provided in solution. Components of anextracellular matrix can include laminin, collagen and fibronectin.

The term “extracellular matrix component”, as used herein, can include amember selected from laminin, collagen, fibronectin and elastin.

The term “electroprocessing” shall be defined broadly to include allmethods of electrospinning, electrospraying, electroaerosoling, andelectrosputtering of materials, combinations of two or more suchmethods, and any other method wherein materials are streamed, sprayed,sputtered or dripped across an electric field and toward a target. Theelectroprocessed material can be electroprocessed from one or moregrounded reservoirs in the direction of a charged substrate or fromcharged reservoirs toward a grounded target. The term electroprocessingis not limited to the specific examples set forth herein, and itincludes any means of using an electrical field for depositing amaterial on a target.

As used herein, the term “electrospinning,” also known as “electrostaticspinning,” includes various processes for forming polymeric fibersincluding nanofibers and microfibers by expressing a liquid polymericformulation through a capillary, syringe or similar implement (referredto herein as a flow tube) under the influence of an electrostatic fieldand collecting the so-formed fibers on a target.

“Electroaerosoling” means a process in which droplets are formed from asolution or melt by streaming an electrically charged polymer solutionor melt through an orifice.

A “growth environment” is an environment in which stem cells willproliferate in vitro. Features of the environment include the medium inwhich the cells are cultured, and a supporting structure (such as asubstrate on a solid surface) if present.

“Growth factor” refers to a substance that is effective to promote thegrowth of cells. Growth factors include, but are not limited to, basicfibroblast growth factor (bFGF), acidic fibroblast growth factor (aFGF),epidermal growth factor (EGF), insulin-like growth factor-I (IGF-T),insulin-like growth factor-II (IGF-II), platelet-derived growthfactor-AB (PDGF), vascular endothelial cell growth factor (VEGF),activin-A, bone morphogenic proteins (BMPs), insulin, cytokines,chemokines, morphogens, neutralizing antibodies, other proteins, andsmall molecules.

“Hydrogel” refers to a water-insoluble and water-swellable cross-linkedpolymer that is capable of absorbing at least 3 times, preferably atleast 10 times, its own weight of a liquid. “Hydrogel” can also refer toa “thermo-responsive polymer” as used herein.

As used herein, “scaffold” refers to a structure, comprising abiocompatible material, that provides a surface suitable for adherenceand proliferation of cells. A scaffold may further provide mechanicalstability and support. A scaffold may be in a particular shape or formso as to influence or delimit a three-dimensional shape or form assumedby a population of proliferating cells. Such shapes or forms include,but are not limited to, films (e.g. a form with two-dimensionssubstantially greater than the third dimension), ribbons, cords, sheets,flat discs, cylinders, spheres, 3-dimensional amorphous shapes, etc.

The term “isolated” refers to a material that is substantially oressentially free from components, which are used to produce thematerial. The lower end of the range of purity for the compositions isabout 60%, about 70% or about 80% and the upper end of the range ofpurity is about 70%, about 80%, about 90% or more than about 90%.

As used here, “biocompatible” refers to any material, which, whenimplanted in a mammal, does not provoke an adverse response in themammal. A biocompatible material, when introduced into an individual, isnot toxic or injurious to that individual, nor does it induceimmunological rejection of the material in the mammal.

As used herein, a “graft” refers to a cell, tissue or organ that isimplanted into an individual, typically to replace, correct or otherwiseovercome a defect. A graft may further comprise a scaffold. The tissueor organ may consist of cells that originate from the same individual;this graft is referred to herein by the following interchangeable terms:“autograft”, “autologous transplant”, “autologous implant” and“autologous graft”. A graft comprising cells from a geneticallydifferent individual of the same species is referred to herein by thefollowing interchangeable terms: “allograft”, “allogeneic transplant”,“allogeneic implant” and “allogeneic graft”. A graft from an individualto his identical twin is referred to herein as an “isograft”, a“syngeneic transplant”, a “syngeneic implant” or a “syngeneic graft”. A“xenograft”, “xenogeneic transplant” or “xenogeneic implant” refers to agraft from one individual to another of a different species.

As used herein, the terms “tissue grafting” and “tissue reconstructing”both refer to implanting a graft into an individual to treat oralleviate a tissue defect, such as a lung defect or a soft tissuedefect.

As used herein, to “alleviate” a disease, defect, disorder or conditionmeans reducing the severity of one or more symptoms of the disease,defect, disorder or condition.

As used herein, to “treat” means reducing the frequency with whichsymptoms of a disease, defect, disorder, or adverse condition, and thelike, are experienced by a patient.

As used herein, a “therapeutically effective amount” is the amount of acomposition of the invention sufficient to provide a beneficial effectto the individual to whom the composition is administered.

As used herein, the term “growth medium” is meant to refer to a culturemedium that promotes growth of cells. A growth medium will generallycontain animal serum. In some instances, the growth medium may notcontain animal serum.

“Differentiation medium” is used herein to refer to a cell growth mediumcomprising an additive or a lack of an additive such that a stem cell,fetal pulmonary cell or other such progenitor cell, that is not fullydifferentiated, develops into a cell with some or all of thecharacteristics of a differentiated cell when incubated in the medium.

An “isolated cell” refers to a cell which has been separated from othercomponents and/or cells which naturally accompany the isolated cell in atissue or mammal.

As used herein, a “substantially purified” cell is a cell that isessentially free of other cell types. Thus, a substantially purifiedcell refers to a cell which has been purified from other cell types withwhich it is normally associated in its naturally-occurring state.

“Expandability” is used herein to refer to the capacity of a cell toproliferate, for example, to expand in number or, in the case of apopulation of cells, to undergo population doublings.

“Proliferation” is used herein to refer to the reproduction ormultiplication of similar forms, especially of cells. That is,proliferation encompasses production of a greater number of cells, andcan be measured by, among other things, simply counting the numbers ofcells, measuring incorporation of ³H-thymidine into the cell, and thelike.

As used herein, “tissue engineering” refers to the process of generatingtissues ex vivo for use in tissue replacement or reconstruction. Tissueengineering is an example of “regenerative medicine,” which encompassesapproaches to the repair or replacement of tissues and organs byincorporation of cells, gene or other biological building blocks, alongwith bioengineered materials and technologies.

As used herein “endogenous” refers to any material from or producedinside an organism, cell or system.

“Exogenous” refers to any material introduced into or produced outsidean organism, cell, or system.

“Encoding” refers to the inherent property of specific sequences ofnucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, toserve as templates for synthesis of other polymers and macromolecules inbiological processes having either a defined sequence of nucleotides(i.e., rRNA, tRNA and mRNA) or a defined sequence of amino acids and thebiological properties resulting therefrom. Thus, a gene encodes aprotein if transcription and translation of mRNA corresponding to thatgene produces the protein in a cell or other biological system. Both thecoding strand, the nucleotide sequence of which is identical to the mRNAsequence and is usually provided in sequence listings, and thenon-coding strand, used as the template for transcription of a gene orcDNA, can be referred to as encoding the protein or other product ofthat gene or cDNA.

Unless otherwise specified, a “nucleotide sequence encoding an aminoacid sequence” includes all nucleotide sequences that are degenerateversions of each other and that encode the same amino acid sequence.Nucleotide sequences that encode proteins and RNA may include introns.

An “isolated nucleic acid” refers to a nucleic acid segment or fragmentwhich has been separated from sequences which flank it in anaturally-occurring state, i.e., a DNA fragment which has been removedfrom the sequences which are normally adjacent to the fragment, i.e.,the sequences adjacent to the fragment in a genome in which it naturallyoccurs. The term also applies to nucleic acids which have beensubstantially purified from other components which naturally accompanythe nucleic acid, i.e., RNA or DNA or proteins, which naturallyaccompany it in the cell. The term therefore includes, for example, arecombinant DNA which is incorporated into a vector, into anautonomously replicating plasmid or virus, or into the genomic DNA of aprokaryote or eukaryote, or which exists as a separate molecule (i.e.,as a cDNA or a genomic or cDNA fragment produced by PCR or restrictionenzyme digestion) independent of other sequences. It also includes arecombinant DNA which is part of a hybrid gene encoding additionalpolypeptide sequence.

In the context of the present invention, the following abbreviations forthe commonly occurring nucleic acid bases are used. “A” refers toadenosine, “C” refers to cytosine, “G” refers to guanosine, “T” refersto thymidine, and “U” refers to uridine.

The phrase “under transcriptional control” or “operatively linked” asused herein means that the promoter is in the correct location andorientation in relation to the polynucleotides to control RNA polymeraseinitiation and expression of the polynucleotides.

As used herein, the term “promoter/regulatory sequence” means a nucleicacid sequence which is required for expression of a gene productoperably linked to the promoter/regulatory sequence. In some instances,this sequence may be the core promoter sequence and in other instances,this sequence may also include an enhancer sequence and other regulatoryelements which are required for expression of the gene product. Thepromoter/regulatory sequence may, for example, be one which expressesthe gene product in a tissue specific manner.

A “constitutive” promoter is a nucleotide sequence which, when operablylinked with a polynucleotide which encodes or specifies a gene product,causes the gene product to be produced in a cell under most or allphysiological conditions of the cell.

An “inducible” promoter is a nucleotide sequence which, when operablylinked with a polynucleotide which encodes or specifies a gene product,causes the gene product to be produced in a cell substantially only whenan inducer which corresponds to the promoter is present in the cell.

A “tissue-specific” promoter is a nucleotide sequence which, whenoperably linked with a polynucleotide which encodes or specifies a geneproduct, causes the gene product to be produced in a cell substantiallyonly if the cell is a cell of the tissue type corresponding to thepromoter.

A “vector” is a composition of matter which comprises an isolatednucleic acid and which can be used to deliver the isolated nucleic acidto the interior of a cell. Numerous vectors are known in the artincluding, but not limited to, linear polynucleotides, polynucleotidesassociated with ionic or amphiphilic compounds, plasmids, and viruses.Thus, the term “vector” includes an autonomously replicating plasmid ora virus. The term should also be construed to include non-plasmid andnon-viral compounds which facilitate transfer of nucleic acid intocells, such as, for example, polylysine compounds, liposomes, and thelike. Examples of viral vectors include, but are not limited to,adenoviral vectors, adeno-associated virus vectors, retroviral vectors,and the like.

“Expression vector” refers to a vector comprising a recombinantpolynucleotide comprising expression control sequences operativelylinked to a nucleotide sequence to be expressed. An expression vectorcomprises sufficient cis-acting elements for expression; other elementsfor expression can be supplied by the host cell or in an in vitroexpression system. Expression vectors include all those known in theart, such as cosmids, plasmids (i.e., naked or contained in liposomes)and viruses that incorporate the recombinant polynucleotide.

The term “patient” as used herein includes human and veterinarysubjects.

As used herein, “wound healing” is intended to include all disorderscharacterized by any disease, disorder, syndrome, anomaly, pathology, orabnormal condition of the skin and/or underlying connective tissue,e.g., skin wounds following surgery, skin abrasions caused my mechanicaltrauma, caustic agents or burns, cornea following cataract surgery orcorneal transplants, mucosal epithelium wounds following infection ordrug therapy (e.g., respiratory, gastrointestinal, genitourinary,mammary, oral cavity, ocular tissue, liver and kidney), diabetic wounds,skin wounds following grafting, and regrowth of blood vessels followingangioplasty.

DESCRIPTION OF THE INVENTION

The present invention provides a method of using plant-derived proteinsas a source of biomaterials for tissue engineering purposes. In oneaspect, the invention includes a scaffold produced from a plant product,wherein the scaffold is able to support the growth of animal cells. Inanother aspect, the scaffold mimics natural extracellular matrix (ECM).

The invention includes the use of any proteinaceous products obtainedfrom alimentary plants. Non-limiting examples of alimentary plantsinclude, but are not limited to corn, wheat, potato, sorghums, tapioca,rice, arrow root, sago, soybean, pea, sunflower, peanut, gelatin. Theinvention provides a means of generating biomaterials from products andthe biomaterials can be used as a scaffold in tissue engineering, drugdelivery, drug discovery, therapy, and other research purposes.

Composition

The invention is based on the discovery that plant products such as soyprotein and corn zein can be used to produce a scaffold capable ofsupporting the growth of cells and therefore provides an alternativebiodegradable composition for biomedical applications. However, theinvention should not be limited to only soy and corn. Rather, theinvention includes a novel method of using any natural product such asproteins obtained from alimentary products.

The possibility to modify both the chemistry and the morphology of thenatural alimentary materials (i.e., materials obtained from analimentary plant) are also claimed in the present invention offering aseries of approaches to make these natural alimentary materials suitablefor many biomedical applications. These modifications aim to modulatethe degradation time of the material varying its porosity and surfacechemistry as well as to improve the material biocompatibility andmechanical properties.

By way of example, the following section describes a soybean-basedscaffold. However, the invention should not be construed to be limitedto a soybean-based scaffold. As used herein, the term “soy material” isdefined as a material derived from soybeans. The term “soybean” refersto the species Glycine max, Glycine soja, or any species that issexually cross compatible with Glycine max.

The term “soy protein isolate” as used herein is used in the senseconventional to the soy protein industry. For example, a soy proteinisolate is a soy material having a protein content of at least 90% soyprotein on a moisture free basis. “Isolated soy protein”, as used in theart, has the same meaning as “soy protein isolate” as used herein and asused in the art. A soy protein isolate is formed from soybeans byremoving the hull and germ of the soybean from the cotyledon, flaking orgrinding the cotyledon and removing oil from the flaked or groundcotyledon, separating the soy protein and carbohydrates of the cotyledonfrom the cotyledon fiber, and subsequently separating the soy proteinfrom the carbohydrates.

In one embodiment, the soy-based composition comprises a fibrousmaterial containing soy protein and soy cotyledon fiber. The fibrousmaterial generally comprises a defatted soy protein material and soycotyledon fiber. The fibrous material is produced by extruding the soyprotein material and soy cotyledon fiber. The fibrous material has amoisture content of from 6% to 80%. Moisture conditions employed inproducing the fibrous material are low moisture fibrous material (6% to35%) and high moisture fibrous material (50% to 80%). Additionalingredients may be extruded with the soy protein material and the soycotyledon fiber such as wheat gluten and starch.

The soy protein isolate should not be a highly hydrolyzed soy proteinisolate having a low molecular weight distribution since highlyhydrolyzed soy protein isolates lack the protein chain length toproperly form protein fibers in the process. Highly hydrolyzed soyprotein isolates, however, may be used in combination with other soyprotein isolates provided that the highly hydrolyzed soy protein isolatecontent of the combined soy protein isolates is less than 40% of thecombined soy protein isolates, by weight.

The soy protein isolate utilized should have a water holding capacitysufficient to enable the protein in the isolate to form fibers uponextrusion. The water holding capacity of the soy protein isolate is ameasure of the amount of swelling the protein undergoes when hydrated.The swelling of the protein should be sufficient to enable the proteinto form intermolecular contacts to permit fiber formation to occur. Thesoy protein isolate used in the process of the invention preferably hasa water holding capacity of at least 4.0 grams of water per gram of soyprotein isolate (as is) at pH 7.0, and more preferably has a waterholding capacity of at least 5.0 grams of water per gram of soy proteinisolate (as is) at pH 7.0. The water holding capacity is determined byusing the centrifuge method.

Non-highly hydrolyzed soy protein isolates having a water holdingcapacity of at least 4.0 grams of water per gram of soy protein isolatethat are useful in the present invention are commercially available.

Soy protein isolates useful in the fibrous material may be produced fromsoybeans according to conventional processes in the soy proteinmanufacturing industry. Exemplary of such a process, whole soybeans areinitially detrashed, cracked, dehulled, degermed, and defatted accordingto conventional processes to form soy flakes, soy flour, soy grits, orsoy meal. The soybeans may be detrashed by passing the soybeans througha magnetic separator to remove iron, steel, and other magneticallysusceptible objects, followed by shaking the soybeans on progressivelysmaller meshed screens to remove soil residues, pods, stems, weed seeds,undersized beans, and other trash. The detrashed soybeans may be crackedby passing the soybeans through cracking rolls. Cracking rolls arespiral-cut corrugated cylinders which loosen the hull as the soybeanspass through the rolls and crack the soybean material into severalpieces. The cracked soybeans may then be dehulled by aspiration. Thedehulled soybeans are degermed by shaking the dehulled soybeans on ascreen of sufficiently small mesh size to remove the small sized germand retain the larger cotyledons of the beans. The cotyledons are thenflaked by passing the cotyledons through a flaking roll. The flakedcotyledons are defatted by extracting oil from the flakes bymechanically expelling the oil from the flakes or by contacting theflakes with hexane or other suitable lipophilic/hydrophobic solvent. Thedefatted flakes may be ground to form a soy flour, a soy grit, or a soymeal, if desired.

The defatted soy flakes, soy flour, soy grits, or soy meal is/are thenextracted with an aqueous alkaline solution, typically a dilute aqueoussodium hydroxide solution having a pH of from 7.5 to 11.0, to extractprotein soluble in an aqueous alkaline solution from insolubles. Theinsolubles are soy cotyledon fiber which is composed primarily ofinsoluble carbohydrates. An aqueous alkaline extract containing thesoluble protein is subsequently separated from the insolubles, and theextract is then treated with an acid to lower the pH of the extract toaround the isoelectric point of the soy protein, preferably to a pH offrom 4.0 to 5.0, and most preferably to a pH of from 4.4 to 4.6. The soyprotein precipitates from the acidified extract due to the protein'slack of solubility in an aqueous solution at or near its isoelectricpoint. The precipitated protein curd is then separated from theremaining extract. The separated protein may be washed with water toremove residual soluble carbohydrates and ash from the protein material.The separated protein is then dried using conventional drying means suchas spray drying or tunnel drying to form a soy protein isolate.

Soy protein concentrate may be blended with the soy protein isolate tosubstitute for a portion of the soy protein isolate as a source of soyprotein. Soy protein isolates, in general, have higher water holdingcapacity and form better fibers than soy protein concentrates.Therefore, the amount of soy protein concentrate substituted for soyprotein isolate should be limited to an amount that will permitsignificant fiber formation in the extrudate. Preferably, if a soyprotein concentrate is substituted for a portion of the soy proteinisolate, the soy protein concentrate is substituted for up to 40% of thesoy protein isolate by weight, at most, and more preferably issubstituted for up to 30% of the soy protein isolate by weight.

Soy protein concentrates useful in the fibrous material are commerciallyavailable. Soy protein concentrates useful in the present invention mayalso be produced from soybeans according to conventional processes inthe soy protein manufacturing industry. For example, defatted soyflakes, soy flour, soy grits, or soy meal produced as described abovemay be washed with aqueous ethanol (preferably 60% to 80% aqueousethanol) to remove soluble carbohydrates from the soy protein and soyfiber. The soy protein and soy fiber containing material is subsequentlydried to produce the soy protein concentrate. Alternatively, thedefatted soy flakes, soy flour, soy grits, or soy meal may be washedwith an aqueous acidic wash having a pH of from 4.3 to 4.8 to removesoluble carbohydrates from the soy protein and soy fiber. The soyprotein and soy fiber containing material is subsequently dried toproduce the soy protein concentrate.

The soy cotyledon fiber utilized in the fibrous material shouldeffectively bind water when the mixture of soy protein material and soycotyledon fiber are co-extruded. By binding water, the soy cotyledonfiber induces a viscosity gradient across the extrudate as the extrudateis extruded through a cooling die, thereby promoting the formation ofprotein fibers. To effectively bind water for the purposes of theprocess of the present invention, the soy cotyledon fiber should have awater holding capacity of at least 5.50 grams of water per gram of soycotyledon fiber, and preferably the soy cotyledon fiber has a waterholding capacity of at least 6.0 grams of water per gram of soycotyledon fiber. It is also preferable that the soy cotyledon fiber hasa water holding capacity of at most 8.0 grams of water per gram of soycotyledon fiber.

The soy cotyledon fiber is a complex carbohydrate and is commerciallyavailable. Soy cotyledon fiber useful in the process of the presentinvention may also be produced according to conventional processes inthe soy processing industry. For example, defatted soy flakes, soyflour, soy grits, or soy meal produced as described above may beextracted with an aqueous alkaline solution as described above withrespect to the production of a soy protein isolate to separate theinsoluble soy cotyledon fiber from the aqueous alkaline soluble soyprotein and carbohydrates. The separated soy cotyledon fiber is thendried, preferably by spray drying, to produce a soy cotyledon fiberproduct. Soy cotyledon fiber is generally present in the fibrousmaterial at from 1% to 8%, preferably at from 1.5% to 7.5% and mostpreferably at from 2% to 5% by weight on a moisture free basis.

The invention provides the use of soybean proteins for the generation ofa biomaterial useful for engineering applications. When deprived fromits oil component, soybean flour is a natural composite mainlyconstituted by proteins and carbohydrates. The production of the soybeanmilk from the ground flour and its processing into cheese of differenttexture by calcium solutions have been largely explored in food industryto provide healthy alimentary products. The disclosure presented hereindemonstrate the production of a soy-based biomaterial that can be usedto support the growth of cells and therefore demonstrate theapplicability of soy in the biomedical field.

The invention provides fibers as well as nanofibrous biocompatiblebiomatrices electrospun from a natural product such as soy. In someinstances, the natural product is blended with a synthetic polymer, suchas poly(ethylene oxide) (PEO) to produce a tissue engineering scaffold.The particular blends provide a unique mix of mechanical and physicalproperties that facilitates cell penetration and proliferation withinthe scaffolds without crosslinking.

Methods of Making a Scaffold

The scaffolds of the invention can be produced in a variety of ways. Inan exemplary embodiment, the scaffold can be produced byelectrospinning. Electrospinning is an atomization process of aconducting fluid which exploits the interactions between anelectrostatic field and the conducting fluid. When an externalelectrostatic field is applied to a conducting fluid (e.g., asemi-dilute polymer solution or a polymer melt), a suspended conicaldroplet is formed, whereby the surface tension of the droplet is inequilibrium with the electric field. Electrostatic atomization occurswhen the electrostatic field is strong enough to overcome the surfacetension of the liquid. The liquid droplet then becomes unstable and atiny jet is ejected from the surface of the droplet. As it reaches agrounded target, the material can be collected as an interconnected webcontaining relatively fine, i.e. small diameter, fibers. The resultingfilms (or membranes) from these small diameter fibers have very largesurface area to volume ratios and small pore sizes. A detaileddescription of electrospinning apparatus is provided in Zong, et al.,2002 Polymer 43: 4403-4412; Rosen et al., 1990 Ann Plast Surg 25:375-87; Kim, K., Biomaterials 2003, 24: 4977-85; Zong, X., 2005Biomaterials 26: 5330-8. After electrospinninng, extrusion and moldingcan be utilized to further fashion the polymers. To modulate fiberorganization into aligned fibrous polymer scaffolds, the use ofpatterned electrodes, wire drum collectors, or post-processing methodssuch as uniaxial stretching has been successful. Zong, X., 2005Biomaterials 26: 5330-8; Katta, P., 2004 Nano Lett 4: 2215-2218; Li, D.,2005 Nano Lett 5: 913-6.

The protein solution comprising a product derived from a plant can beproduced in one of several ways. One method involves dissolving thesubsequent plant product in an appropriate solvent. This process can beaccomplished in a syringe assembly or it can be subsequently loaded intoa syringe assembly. Another method involves purchasing commerciallyavailable polymer solutions or commercially available polymers anddissolving them to create polymer solutions. For example, poly(ethyleneoxide) (PEO) can be purchased from Sigma (Sigma, St. Louis, Mo.),poly-L-lactide (PLLA) can be purchased from DuPont (Wilmington, Del.),poly(lactide-co-glycolide) can be purchased from Ethicon (Somerville,N.J.). Additional polymer scaffold components of the invention, such ascells and biomolecules, are also commercially available from suppliers.

The protein solution comprising a product derived from a plant used toform scaffold is first dissolved in a solvent. The solvent can be anysolvent which is capable of dissolving the plant product and/or subunitsthereof. Typical solvents include a solvent selected from N,N-Dimethylformamide (DMF), tetrahydrofuran (THF), methylene chloride, dioxane,ethanol, hexafluoroisopropanol (HFIP), chloroform,1,1,1,3,3,3-hexafluoro-2-propanol (HFP), glacial acetic acid, water, andcombinations thereof.

The protein solution can optionally contain a salt which creates anexcess charge effect to facilitate the electrospinning process. Examplesof suitable salts include NaCl, KH₂PO₄, K₂HPO₄, KIO₃, KCl, MgSO₄, MgCl₂,NaHCO₃, CaCl₂ or mixtures of these salts.

The protein solution forming the conducting fluid preferably has aprotein concentration in the range of about 1 to about 80 wt %, morepreferably about 8 to about 60 wt %.

The electric field created in the electrospinning process preferably isin the range of about 5 to about 100 kilovolts (kV), more preferablyabout 10 to about 50 kV. The feed rate of the conducting fluid to thespinneret (or electrode) preferably is in the range of about 0.1 toabout 1000 microliters/min, more preferably about 1 to about 250microliters/min.

The single or multiple spinnerets sit on a platform which is capable ofbeing adjusted, varying the distance between the platform and thegrounded collector substrate. The distance can be any distance whichallows the solvent to essentially completely evaporate prior to thecontact of the polymer with the grounded collector substrate. In anexemplary embodiment, this distance can vary from 1 cm to 25 cm.Increasing the distance between the grounded collector substrate and theplatform generally produces thinner fibers.

In electrospinning cases where a rotating mandrel is required, themandrel is mechanically attached to a motor, often through a drillchuck. In an exemplary embodiment, the motor rotates the mandrel at aspeed of between about 1 revolution per minute (rpm) to about 500 rpm.In an exemplary embodiment, the motor rotation speed of between about200 rpm to about 500 rpm. In another exemplary embodiment, the motorrotation speed of between about 1 rpm to about 100 rpm.

Additional embodiments or modifications to the electrospinning processand apparatus are described herein.

The invention also includes combinations of natural materials,combinations of synthetic materials, and combinations of both naturaland synthetic materials. Examples of combinations include, but are notlimited to: blends of different types of collagen (e.g. Type I with TypeII, Type I with Type III, Type II with Type III, etc.); blends of one ormore types of collagen with fibrinogen, thrombin, elastin, PGA, PLA, andpolydioxanone; and blends of fibrinogen with one or more types ofcollagen, thrombin, elastin, PGA, PLA, and polydioxanone.

The electroprocessed material of the present invention can result fromthe electroprocessing of natural materials, synthetic materials, orcombinations thereof. Examples include but are not limited to aminoacids, peptides, denatured peptides such as gelatin from denaturedcollagen, polypeptides, proteins, carbohydrates, lipids, nucleic acids,glycoproteins, lipoproteins, glycolipids, glycosaminoglycans, andproteoglycans.

Some preferred materials to be electroprocessed are naturally occurringextracellular matrix materials and blends of naturally occurringextracellular matrix materials, including but not limited to collagen,fibrin, fibrinogen, thrombin, elastin, laminin, fibronectin, hyaluronicacid, chondroitin 4-sulfate, chondroitin 6-sulfate, dermatan sulfate,heparin sulfate, heparin, and keratan sulfate, and proteoglycans.Especially preferred materials for electroprocessing include collagen,fibrin, fibrinogen, thrombin, fibronectin, and combinations thereof.Some collagens that are used include but are not limited to collagentypes I, II, III, IV, V, VI, VII, VIII, IX, X, XI, XII, XIII, XIV, XV,XVI, XVII, XVIII, and XIX. Some preferred collagens include types I, II,and III. These proteins may be in any form, including but not limited tonative and denatured forms. Other preferred materials forelectroprocessing are carbohydrates such as polysaccharides (e.g.cellulose and its derivatives), chitin, chitosan, alginic acids, andalginates such as calcium alginate and sodium alginate. These materialsmay be isolated from plant products, humans or other organisms or cellsor synthetically manufactured. Some especially preferred naturalmaterials for electroprocessing are collagen, fibrinogen, thrombin,fibrin, fibronectin, and combinations thereof. Also included are crudeextracts of tissue, extracellular matrix material, extracts ofnon-natural tissue, or extracellular matrix materials (i.e. extracts ofcancerous tissue), alone or in combination. Extracts of biologicalmaterials, including but are not limited to cells, tissues, organs, andtumors may also be electroprocessed.

Collagen and fibrinogen can each been electrospun to produce fibershaving repeating, band patterns along the length of the fibers. Thesepatterns are observable, for example with transmission electronmicroscopy, and are typical of those produced by natural processes. Insome embodiments, the banded pattern observed in electrospun collagenfibers is the same as that produced by cells in vivo. In someembodiments, the banding pattern in electrospun fibrinogen is the sameas that of fibrinogen found in normal clots formed in vivo. While notwishing to be bound by any particular theory, it is believed that thebanding apparent along natural collagen fibers results from the helicalpattern of the protein chains in the collagen, while the banding infibrinogen in vivo results from close packing of individual fibrinmolecules in a stacked configuration. In some of these embodiments, thecompositions are composed of fibrous webs rather than networkscharacteristic of fibrin clots. Further, in some embodiments,electroprocessed fibrinogen is not soluble in water, unlike nativefibrinogen.

The invention includes all natural or natural-synthetic hybridcompositions that result from the electroprocessing of any material.Materials that change in composition or structure before, during, orafter electroprocessing are within the scope of the invention.

It is to be understood that these electroprocessed materials may becombined with other materials and/or substances in forming thecompositions of the present invention. For example, in some embodimentsan electroprocessed peptide is combined with an adjuvant to enhanceimmunogenicity when implanted subcutaneously. Electroprocessed materialsin some embodiments are prepared at very basic or acidic pHs (forexample, by electroprocessing from a solution having a specific pH) toaccomplish the same effect. As another example, an electroprocessedmatrix, containing cells, may be combined with an electroprocessedbiologically compatible polymer and growth factors to stimulate growthand division of the cells in the electroprocessed matrix.

Synthetic materials electroprocessed for use in the scaffold include anymaterials prepared through any method of artificial synthesis,processing, isolation, or manufacture. The synthetic materials arepreferably biologically compatible for administration in vivo or invitro. Such polymers include but are not limited to the following:poly(urethanes), poly(siloxanes) or silicones, poly(ethylene),poly(vinyl pyrrolidone), poly(2-hydroxy ethyl methacrylate),poly(N-vinyl pyrrolidone), poly(methyl methacrylate), poly(vinylalcohol), poly(acrylic acid), polyacrylamide, poly(ethylene-co-vinylacetate), poly(ethylene glycol), poly(methacrylic acid), polylactic acid(PLA), polyglycolic acids (PGA), poly(lactide-co-glycolides) (PLGA),nylons, polyamides, polyanhydrides, poly(ethylene-co-vinyl alcohol)(EVOH), polycaprolactone, poly(vinyl acetate) (PVA), polyvinylhydroxide,poly(ethylene oxide) (PEO) and polyorthoesters or any other similarsynthetic polymers that may be developed that are biologicallycompatible. Some preferred synthetic materials include PLA, PGA,copolymers of PLA and PGA, polycaprolactone, poly(ethylene-co-vinylacetate), EVOH, PVA, and PEO. Polymers with cationic moieties are alsopreferred in some embodiments. Examples of such polymers include, butare not limited to, poly(allyl amine), poly(ethylene imine),poly(lysine), and poly(arginine). The polymers may have any molecularstructure including, but not limited to, linear, branched, graft, block,star, comb and dendrimer structures. Matrices can be formed ofelectrospun fibers, electroaerosol, electrosprayed, or electrosputtereddroplets, electroprocessed powders or particles, or a combination of theforegoing.

By selecting different natural and synthetic materials, or combinationsthereof, many characteristics of the scaffold are manipulated. Theproperties of the matrix comprised of electroprocessed material and asubstance may be adjusted. In addition, selection of materials forelectroprocessing can affect the permanency of an implanted matrix. Forexample, many matrices made by electroprocessing fibrinogen or fibrinmay degrade more rapidly while many matrices made of collagen are moredurable and many other matrices made by electroprocessing materials aremore durable still. Thus, for example, incorporation of durablesynthetic polymers (e.g. PLA, PGA) increase the durability andstructural strength of matrices electroprocessed from solutions offibrinogen in some embodiments. Use of matrices made byelectroprocessing natural materials such as proteins derived from corn,wheat, potato, sorghums, tapioca, rice, arrow root, sago, soybean, pea,sunflower, peanut, gelatin, and the like also minimize rejection orimmunological response to an implanted matrix. Accordingly, selection ofmaterials for electroprocessing and use in substance delivery isinfluenced by the desired use.

In one embodiment, a skin patch of material electroprocessed fromfibrin, fibrinogen, fibronectin, collagen or a combination of one ormore of these is combined with healing promoters, analgesics and oranesthetics and anti-rejection substances and applied to the skin andmay subsequently dissolve into the skin. In another embodiment, anelectroprocessed implant for delivery to bone may be constructed ofmaterials useful for promoting bone growth, osteoblasts andhydroxyapatite, and may be designed to endure for a prolonged period oftime. In embodiments in which the matrix contains substances that are tobe released from the matrix, incorporating electroprocessed syntheticcomponents, such as biocompatible substances, can modulate the releaseof substances from an electroprocessed composition. For example, layeredor laminate structures can be used to control the substance releaseprofile. Unlayered structures can also be used, in which case therelease is controlled by the relative stability of each component of theconstruct. For example, layered structures composed of alternatingelectroprocessed materials are prepared by sequentiallyelectroprocessing different materials onto a target. The outer layersare, for example, tailored to dissolve faster or slower than the innerlayers. Multiple agents can be delivered by this method, optionally atdifferent release rates. Layers can be tailored to provide a complex,multi-kinetic release profile of a single agent over time. Usingcombinations of the foregoing provides for release of multiplesubstances released, each with its own profile. Complex profiles arepossible.

Natural components such as biocompatible substances can be used tomodulate the release of electroprocessed materials or of substances froman electroprocessed composition. For example, a drug or series of drugsor other materials or substances to be released in a controlled fashioncan be electroprocessed into a series of layers. In one embodiment, onelayer is composed of electroprocessed fibrinogen plus a drug, the nextlayer PLA plus a drug, a third layer is composed of polycaprolactoneplus a drug. The layered construct can be implanted, and as thesuccessive layers dissolve or break down, the drug (or drugs) isreleased in turn as each successive layer erodes. In some embodiments,unlayered structures are used, and release is controlled by the relativestability of each component of the construct.

In some embodiments, the electroprocessed material itself may provide atherapeutic effect. Non-limiting examples of a material that has atherapeutic effect is electroprocessed fibrinogen, thrombin, fibrin, orcombinations thereof. For example, thrombin converts fibrinogen tofibrin. Fibrin assists in arrest of bleeding (hemostasis). Fibrin is acomponent of the provisional matrix that is laid down during the earlystages of healing and may also promote the growth of vasculature inadjacent region. In many ways fibrin is a natural healing promoter. Insome embodiments, electroprocessed fibrinogen also assists in healing.When placed in contact with a wound of a patient, such anelectroprocessed material provides the same healing properties asfibrin.

Method for Forming Matrices or Scaffolds

The biocompatible scaffold may be shaped using methods such as, forexample, solvent casting, compression molding, filament drawing,meshing, leaching, weaving, foaming, electrospinning and coating. Insolvent casting, a solution of one or more proteins in an appropriatesolvent, is cast as a branching pattern relief structure. After solventevaporation, a thin film is obtained. In compression molding, a polymeris pressed at pressures up to 30,000 pounds per square inch into anappropriate pattern. Filament drawing involves drawing from the moltenpolymer and meshing involves forming a mesh by compressing fibers into afelt-like material. In leaching, a solution containing two materials isspread into a shape close to the final form of the artificial organ.Next a solvent is used to dissolve away one of the components, resultingin pore formation. (See U.S. Pat. No. 5,514,378 to Mikos).

The scaffold may be shaped into any number of desirable configurationsto satisfy any number of overall system, geometry or space restrictions.For example, in the use of the scaffold for bladder, urethra, valve, orblood vessel reconstruction, the matrix or scaffold may be shaped toconform to the dimensions and shapes of the whole or a part of thetissue. The scaffold may be shaped in different sizes and shapes toconform to the organs of differently sized patients. For bladders, thescaffold should be shaped such that after its biodegradation, theresulting reconstructed bladder may be collapsible when empty in afashion similar to a natural bladder. The matrix or scaffold may also beshaped in other fashions to accommodate the special needs of thepatient.

In one embodiment, the scaffolds are seeded with one or more populationsof cells to form an artificial organ construct. The artificial organconstruct can be autologous, where the cell populations are derived fromthe subject's own tissue, or allogenic, where the cell populations arederived from another subject within the same species as the patient. Theartificial organ construct can also be xenogenic, where the differentcell populations are derived form a mammalian species that is differentfrom the subject. For example the cells can be derived from organs ofmammals such as humans, monkeys, dogs, cats, mice, rats, cows, horses,pigs, goats and sheep.

Cells can be isolated from a number of sources, including, for example,biopsies from living subjects and whole-organ recover from cadavers. Theisolated cells are preferably autologous cells, obtained by biopsy fromthe subject intended to be the recipient. For example, a biopsy ofskeletal muscle from the arm, forearm, or lower extremities, or smoothmuscle from the area treated with local anesthetic with a small amountof lidocaine injected subcutaneously, and expanded in culture. Thebiopsy can be obtained using a biopsy needle, a rapid action needlewhich makes the procedure quick and simple.

Cells may be isolated using techniques known to those skilled in theart. For example, the tissue or organ can be disaggregated mechanicallyand/or treated with digestive enzymes and/or chelating agents thatweaken the connections between neighboring cells making it possible todisperse the tissue into a suspension of individual cells withoutappreciable cell breakage. Enzymatic dissociation can be accomplished bymincing the tissue and treating the minced tissue with any of a numberof digestive enzymes either alone or in combination. These include butare not limited to trypsin, chymotrypsin, collagenase, elastase, and/orhyaluronidase, DNase, pronase and dispase. Mechanical disruption canalso be accomplished by a number of methods including, but not limitedto, scraping the surface of the organ, the use of grinders, blenders,sieves, homogenizers, pressure cells, or insonicators.

Preferred cell types include, but are not limited to, urothelial cells,mesenchymal cells, especially smooth or skeletal muscle cells, myocytes(muscle stem cells), fibroblasts, chondrocytes, adipocytes,fibromyoblasts, and ectodermal cells, including ductile and skin cells,hepotocytes, Islet cells, cells present in the intestine, and otherparenchymal cells, osteoblasts and other cells forming bone orcartilage. In some cases, it may also be desirable to include nervecells. In other cases, it mal be desirable to include stem cells.

Once the tissue has been reduced to a suspension of individual cells,the suspension can be fractionated into subpopulations from which thecells elements can be obtained. This also may be accomplished usingstandard techniques for cell separation including, but not limited to,cloning and selection of specific cell types, selective destruction ofunwanted cells (negative selection), separation based upon differentialcell agglutinability in the mixed population, freeze-thaw procedures,differential adherence properties of the cells in the mixed population,filtration, conventional and zonal centrifugation, centrifugalelutriation (counterstreaming centrifugation), unit gravity separation,countercurrent distribution, electrophoresis and fluorescence-activatedcell sorting.

Cell fractionation may also be desirable, for example, when the donorhas diseases such as cancer or metastasis of other tumors to the desiredtissue. A cell population may be sorted to separate malignant cells orother tumor cells from normal noncancerous cells. The normalnoncancerous cells, isolated from one or more sorting techniques, maythen be used for organ reconstruction.

Isolated cells can be cultured in vitro to increase the number of cellsavailable for coating the biocompatible scaffold. The use of allogeniccells, and more preferably autologous cells, is preferred to preventtissue rejection. However, if an immunological response does occur inthe subject after implantation of the artificial organ, the subject maybe treated with immunosuppressive agents such as, cyclosporin or FK506,to reduce the likelihood of rejection. In certain embodiments, chimericcells, or cells from a transgenic animal, can be coated onto thebiocompatible scaffold.

Isolated cells may be transfected prior to coating with geneticmaterial. Useful genetic material may be, for example, genetic sequenceswhich are capable of reducing or eliminating an immune response in thehost. For example, the expression of cell surface antigens such as classI and class II histocompatibility antigens may be suppressed. This mayallow the transplanted cells to have reduced chance of rejection by thehost. In addition, transfection could also be used for gene delivery.

Isolated cells can be normal or genetically engineered to provideadditional or normal function. Methods for genetically engineering cellswith retroviral vectors, polyethylene glycol, or other methods known tothose skilled in the art can be used. These include using expressionvectors which transport and express nucleic acid molecules in the cells.(See Goeddel; Gene Expression Technology: Methods in Enzymology 185,Academic Press, San Diego, Calif. (1990).

Vector DNA is introduced into prokaryotic or cells via conventionaltransformation or transfection techniques. Suitable methods fortransforming or transfecting host cells can be found in Sambrook et al.(Molecular Cloning: A Laboratory Manual, 3nd Edition, Cold Spring HarborLaboratory press (2001)), and other laboratory textbooks.

Seeding of cells onto the matrix or scaffold can be performed accordingto standard methods. For example, the seeding of cells onto polymericsubstrates for use in tissue repair has been reported (see, e.g., Atala,A. et al., J. Urol. 148(2 Pt 2): 658-62 (1992); Atala, A., et al. J.Urol. 150 (2 Pt 2): 608-12 (1993)). Cells grown in culture can betrypsinized to separate the cells, and the separated cells can be seededon the matrix. Alternatively, cells obtained from cell culture can belifted from a culture plate as a cell layer, and the cell layer can bedirectly seeded onto the scaffold without prior separation of the cells.

In a preferred embodiment, in the range of 1 million to 700 50 millioncells are suspended in medium and applied to each square centimeter of asurface of a scaffold. Preferably, between 1 million and 50 millioncells, and more preferably, between 1 million and 10 million cells aresuspended in media and applied to each square centimeter of a surface ofa scaffold. The matrix or scaffold is incubated under standard culturingconditions, such as, for example, 37° C., 5% CO₂, for a period of timeuntil the cells attached. However, it will be appreciated that thedensity of cells seeded onto the scaffold can be varied. For example,greater cell densities promote greater tissue regeneration by the seededcells, while lesser densities may permit relatively greater regenerationof tissue by cells infiltrating the graft from the host. Other seedingtechniques may also be used depending on the matrix or scaffold and thecells. For example, the cells may be applied to the matrix or scaffoldby vacuum filtration. Selection of cell types, and seeding of cells ontoa scaffold, will be routine to one of ordinary skill in the art in lightof the teachings herein.

In one embodiment, the scaffold is seeded with one population of cellsto form an artificial organ construct. In another embodiment, the matrixor scaffold is seeded on two sides with two different populations ofcells. This may be performed by first seeding one side of the matrix orscaffold and then seeding the other side. For example, the scaffold maybe placed with one side on top and seeded. Then the matrix or scaffoldmay be repositioned so that a second side is on top. The second side maythen be seeded with a second population of cells. Alternatively, bothsides of the matrix or scaffold may be seeded at the same time. Forexample, two cell chambers may be positioned on both sides (i.e., asandwich) of the scaffold. The two chambers may be filled with differentcell populations to seed both sides of the matrix or scaffoldsimultaneously. The sandwiched scaffold may be rotated, or flippedfrequently to allow equal attachment opportunity for both cellpopulations. Simultaneous seeding may be preferred when the pores of thematrix or scaffold are sufficiently large for cell passage from one sideto the other side. Seeding the scaffold on both sides simultaneously canreduce the likelihood that the cells would migrate to the opposite side.

In another embodiment, two separate scaffolds may be seeded withdifferent cell populations. After seeding, the two matrices may beattached together to form a single matrix or scaffold with two differentcell populations on the two sides. Attachment of the scaffolds to eachother may be performed using standard procedures such as fibrin glue,liquid co-polymers, sutures and the like.

In order to facilitate cell growth on the scaffold of the presentinvention, the scaffold may be coated with one or more celladhesion-enhancing agents. These agents include but are not limitedcollagen, laminin, and fibronectin. The scaffold may also contain cellscultured on the scaffold to form a target tissue substitute. The targettissue that may be formed using the scaffold of the present inventionmay be an arterial blood vessel, wherein an array of microfibers isarranged to mimic the configuration of elastin in the medial layer of anarterial blood vessel. In the alternative, other cells may be culturedon the scaffold of the present invention. These cells include, but arenot limited to, cells cultured on the scaffold to form a blood vesselsubstitute, epithelial cells cultured on the scaffold to form epithelialtissue, muscle cells cultured on the scaffold to form muscle tissue,endothelial cells cultured on the scaffold to form endothelial tissue,skeletal muscle cells cultured on the scaffold to form skeletal muscletissue, cardiac muscle cells cultured on the scaffold to form cardiacmuscle tissue, collagen fibers cultured on the scaffold to formcartilage, interstitial valvular cells cultured on the scaffold to formvalvular tissue and mixtures thereof.

Therapeutic Application

Grafting of scaffolds to an organ or tissue to be augmented can beperformed according to the methods described in herein or according toart-recognized methods. The matrix or scaffold can be grafted to anorgan or tissue of the subject by suturing the graft material to thetarget organ. Implanting a neo-organ construct for total organreplacement can be performed according to the methods described hereinor according to art-recognized surgical methods. The scaffold is alsouseful for delivery of biologics, enzymes that activate drugs, proteaseinhibitors, and the like.

In one embodiment, the invention includes the use of the natural proteinbased scaffolds as a platform to direct wound healing by the inductionof native skin fibroblasts and keratinocytes to populate the scaffoldsand secrete appropriate matrix components. In some instances, thescaffold can also include desirable cells. For example, the scaffold canincluded cells that have the ability to express angiogenic growthfactors and cytokines, secrete wound healing related cytokines, secretecollagen, and promote wound healing in vivo.

Scaffolds of the invention described can be useful for clinical andpersonal wound care and soft tissue regeneration. In one aspect of theinvention, scaffold is used as a wound dressing or graft for externalskin wounds. In a clinical setting, the scaffold can be used to treatwounds resulting from trauma, burns, ulcers, abrasions, lacerations,surgery, or other damage. Surgeons can use these grafts to cover andprotect the wound area, to temporarily replace lost or damaged skintissue, and to guide new tissue generation and wound healing into thedamaged area. In a clinical setting, the scaffold may be secured to thewound area using sutures, adhesives, or overlaying bandages. Thescaffold may be cut to match the size of the wound, or may overlap thewound edges.

In another aspect of the invention, the scaffold may be tailored forpersonal/home care by combining the sheet with an adhesive backing tocreate a scaffold bandage. An adhesive section can hold the scaffold inplace on a wounded area and can be removed when the fibers degrade orfuse with the tissue. The scaffold sheet may also be secured with aliquid or gel adhesive.

In another aspect of the invention, scaffold sheets can be used as gauzeto absorb fluid and protect large wounds. This scaffold gauze can bewrapped around a wounded area or secured with tape.

In another aspect of the invention, scaffold sheets can be used to treatinternal soft tissue wounds such as wounds in the amniotic sac, ulcersin the gastrointestinal tract or mucous membranes, gingival damage orrecession, internal surgical incisions or biopsies, etc. The scaffoldgrafts can be sutured or adhered into place to fill or cover the damagedtissue area.

The scaffold has numerous characteristics that are useful for woundhealing. First, the polymer scaffolds described herein that includenanofibers are both nano-porous and breathable. They can preventmicrobes and infectious particles from crossing through, but they allowair flow and moisture penetration which are critical in natural woundhealing.

Second, the fibers in this invention are biodegradable, which allows fortemporary wound coverage followed by eventual ingrowth of new tissue.The choice of material for scaffold wound dressings can be determined tomatch the natural tissue characteristics including mechanical strengthand rate of degradation/tissue regeneration.

Third, the scaffolds may be embedded or conjugated with various factorswhich may be released upon degradation. These factors may include, butare not limited to epidermal growth factor (EGF), platelet derivedgrowth factor (PDGF), basic fibroblast growth factor (bFGF),transforming growth factor-β (TGF-β), and tissue inhibitors ofmetalloproteinases (TIMP), which have been shown to be beneficial inwound healing. Additional wound healing factors such as antibiotics,bacteriocides, fungicides, silver-containing agents, analgesics, andnitric oxide releasing compounds can also be incorporated into thescaffold wound dressings or grafts.

Fourth, scaffold grafts for wound healing may be seeded with cells forfaster tissue regeneration and more natural tissue structure. Thesecells may include, but are not limited to fibroblasts, keratinocytes,epithelial cells, endothelial cells, mesenchymal stem cells, and/orembryonic stem cells.

Fifth, the nano-scale architecture of the nanofibrous scaffolds closelymimics that of the extracellular matrix (ECM) of many common softtissues. For example, the nano-scale fibers are structurally similar tocollagen fibrils found in skin and other tissues. This architecture mayprevent scar formation by providing an organized scaffold for cells tomigrate into a wound. In this aspect of the invention, alignment of thescaffold is preferred to keep cells aligned and organized, rather thanallowing them to arrange randomly as in the formation of scar tissue.Aligned scaffolds may be oriented with respect to a given axis of thewound to allow faster tissue ingrowth and wound coverage.

Scaffold alignment can also be used to closely match the architecture ofnatural tissue ECM. This may include fiber alignment in a singledirection, criss-cross alignment in orthogonal directions, or morecomplicated fiber architecture. In this instance of the invention, thescaffold includes multiple layers of fibers with specific fiberorientation in each layer. Similarly, each individual scaffold layer mayalso contain a specific factor or cell type such as the ones listedpreviously. This allows for creation of polymer scaffolds that canclosely match natural tissue architecture and composition. For example,a simple scaffold wound dressing or graft might include a single layerof aligned fibers. On the other hand, a more complex scaffold skin graftmight include multiple aligned fiber sheets layered in a criss-crosspattern with fibroblasts in the bottom sheets and keratinocytes in thetop sheet, as well as bFGF in the bottom sheets and an antimicrobialagent in the top sheet. Other such combinations are possible, dependingon the specific needs of the patient.

In another embodiment, the scaffold can include a therapeutic agent. Thetherapeutic agent can be an anti-tumor agent including but not limitedto a chemotherapeutic agent, an anti-cell proliferation agent or anycombination thereof.

The invention should not limited to any particular chemotherapeuticagent. Rather, any chemotherapeutic agent can be linked to theantibodies of the invention. For example, any conventionalchemotherapeutic agents of the following non-limiting exemplary classesare included in the invention: alkylating agents; nitrosoureas;antimetabolites; antitumor antibiotics; plant alkyloids; taxanes;hormonal agents; and miscellaneous agents.

An anti-cell proliferation agent can further be defined as anapoptosis-inducing agent or a cytotoxic agent. The apoptosis-inducingagent may be a granzyme, a Bcl-2 family member, cytochrome C, a caspase,or a combination thereof. Exemplary granzymes include granzyme A,granzyme B, granzyme C, granzyme D, granzyme E, granzyme F, granzyme G,granzyme H, granzyme I, granzyme J, granzyme K, granzyme L, granzyme M,granzyme N, or a combination thereof. In other specific aspects, theBcl-2 family member is, for example, Bax, Bak, Bcl-Xs, Bad, Bid, Bik,Hrk, Bok, or a combination thereof.

In additional aspects, the caspase is caspase-1, caspase-2, caspase-3,caspase-4, caspase-5, caspase-6, caspase-7, caspase-8, caspase-9,caspase-10, caspase-11, caspase-12, caspase-13, caspase-14, or acombination thereof. In specific aspects, the cytotoxic agent is TNF-α,gelonin, Prodigiosin, a ribosome-inhibiting protein (RIP), Pseudomonasexotoxin, Clostridium difficile Toxin B, Helicobacter pylori VacA,Yersinia enterocolitica YopT, Violacein, diethylenetriaminepentaaceticacid, irofulven, Diptheria Toxin, mitogillin, ricin, botulinum toxin,cholera toxin, saporin 6, or a combination thereof.

The invention also encompasses tissue regeneration applications. Theobjective of the tissue regeneration therapy approach is to deliver highdensities of repair-competent cells (or cells that can become competentwhen influenced by the local environment) to the defect site in a formatthat optimizes both initial wound mechanics and eventual neotissueproduction. The composition of the instant invention is particularlyuseful in methods to alleviate or treat lung tissue defects inindividuals. Advantageously, the composition of the invention providesfor improved lung tissue regeneration. Specifically, the tissueregeneration is achieved more rapidly as a result of the inventivecomposition.

The composition of the invention may be administered to an individual inneed thereof in a wide variety of ways. Preferred modes ofadministration include intravenous, intravascular, intramuscular,subcutaneous, intracerebral, intraperitoneal, soft tissue injection,surgical placement, arthroscopic placement, and percutaneous insertion,e.g. direct injection, cannulation or catheterization. Most preferredmethods result in localized administration of the inventive compositionto the site or sites of tissue defect. Any administration may be asingle application of a composition of invention or multipleapplications. Administrations may be to single site or to more than onesite in the individual to be treated. Multiple administrations may occuressentially at the same time or separated in time.

EXPERIMENTAL EXAMPLES

The invention is further described in detail by reference to thefollowing experimental examples. These examples are provided forpurposes of illustration only, and are not intended to be limitingunless otherwise specified. Thus, the invention should in no way beconstrued as being limited to the following examples, but rather, shouldbe construed to encompass any and all variations which become evident asa result of the teaching provided herein.

Example 1 Alimentary Protein-Based Scaffold

The following experiments were designed to generate biomaterials thatcan be used as scaffolds for biomedical purposes, such as tissueengineering. The results presented herein demonstrate the feasibility ofproducing a scaffold using plant products as the starting material. Thescaffolds were shown to support cell growth.

The materials and methods employed in these experiments are nowdescribed.

Materials and Methods

Preparation of Protein Solutions

Soy protein isolate (SPI) (obtained from Cargill Health and FoodTechnologies, Minneapolis, Minn.) was blended with poly(ethylene oxide)(PEO) (Sigma, St. Louis, Mo.) by first dissolving 0.5% (w/v) PEO in1,1,1,3,3,3-hexafluoro-2-propanol (HFP) (Sigma) and adding appropriatevolumes from this stock solution to 5, 6, 7, and 8% (w/v) SPI in HFPrespectively. Blend solutions were left to stir at least 48 hours beforeelectrospinning to ensure complete dissolution.

Corn zein was dissolved at 35%, 40% and 45% (w/v) in glacial acetic acid(Fisher Scientific) and left to stir at least 24 h beforeelectrospinning.

Electrospinning of Protein Solutions

Fibers were electrospun by using a syringe pump (KD Scientific SingleSyringe Infusion Pump, Fisher) to eject solution from a 3 mL syringethrough an 18-gauge needle at a delivery rate of 0.8 to 1.0 mL/h, airgap distance of 15 cm, and accelerating voltage of 12 kV for SPI/PEOblend solutions. For zein solutions, delivery rate was 0.5 mL/h, air gapdistance was 15 cm, and accelerating voltage was 20 kV. For cell culturespecimens, 15 mm diameter glass coverslips were attached to arectangular aluminum collector and fiber-coated coverslips were detachedfrom the collector. For mats, fibers were collected directly onto thealuminum collector.

Measurement of Fiber Diameters

Glass coverslips coated with electrospun fibers were mounted onto metalstubs with carbon tape and sputter-coated for 30 sec with platinum andpalladium prior to visualization in an environmental scanning electronmicroscope (ESEM, XL-30 Environmental SEM-FEG). Images were taken ofdifferent areas at 2000× original magnification and measurements takenon UTHSCSA ImageTool 3.0 software (n=100 for each specimen).

Dry samples were mounted as spun. To investigate the degree of swellingwith hydration over time, samples of 6% SPI/0.05% PEO and 40% zein wereimmersed in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with10% fetal bovine serum (FBS), 1× phosphate buffered saline (PBS)solution without calcium and magnesium, and tissue culture grade water(Mediatech Inc., Herndon, Va.) separately for 2 h, 24 h and 72 h. Atthese time points, samples were removed with forceps and rinsed fivetimes in fresh tissue culture grade water before placing in ahybridization oven overnight at 37° C.

Characterization of Mechanical Properties

Mats were electrospun and cut into rectangular pieces and tensile testedusing the Instron 5564 in dry and hydrated states. For hydrated state,samples were immersed in DMEM supplemented with 10% FBS for at least 2 hto simulate a physiological situation where proteases would be present.Samples were tested at a gauge length of 15 mm on a 10 N load cell.Crosshead speed was 1 mm/min for dry samples and 10 mm/min for wetsamples. The test was stopped at specimen break or a predeterminedamount of strain, whichever occurred first. Young's modulus wascalculated from the slope of the first linear region of thestress-strain curve.

Cell Culture

Primary human dermal fibroblasts (HDF), were cultured in DMEM with 4.5g/L glucose supplemented with 10% FBS and 2.5% penicillin/streptomycin(10,000 I.U./mL penicillin, 10,000 μg/mL streptomycin solution,Mediatech Inc., Hemdon, Va.). Flasks were kept in sterile incubators at37° C. and 5% CO₂, and culture medium was changed twice per week. Atconfluence, cells were detached from flasks using 0.25% trypsin/2.21 mMEDTA and centrifuged in complete medium prior to counting and using.

Alamar Blue Assay for Metabolic Activity

Fluorescence spectrophotometric reading was calibrated against cellsgrown on tissue culture polystyrene (TCP) inside a 24-well plate todetermine the appropriate seeding density prior to seeding cells ontoscaffolds.

Cells were seeded at a density of 30,000 per well in 200 μL volumes ontoscaffolds in triplicate in a 24-well plate. For each scaffold, one waskept unseeded as a blank to compensate for the effect of scaffoldmaterial on the readings. Cells were also seeded onto scaffoldselectrospun from 20% PLGA and 8% gelatin as synthetic and naturalcontrol materials respectively, and onto glass and TCP as controls forthe entire experiment.

After allowing the cells to attach to the scaffolds for 1 h, medium with10% (v/v) Alamar Blue (BD, Franklin Lakes, N.J.) was added to each wellto a total volume of 0.5 mL. Plates were incubated at 37° C., 5% CO₂ for3 hours before supernatant from each well was transferred to a 96-wellplate in 200 μL volumes in duplicate. Alamar Blue fluorescence was readon a Cytofluor fluorescence spectrophotometer.

Visualization of Cell Morphology on Scaffolds

Morphology of cells was assessed using fluorescence microscopy andenvironmental scanning electron microscopy (ESEM). For fluorescencemicroscopy, cells were fixed in 3.8% paraformaldehyde (Fisher) for 10minutes, washed three times with 1×PBS, and stained with 4 gig/mLHoechst 33258 (bisBenzimide, Sigma) (BBZ) and 2 μg/mL TRITC-conjugatedrhodamine phalloidin (phalloidin-tetramethylrhodamine β isothiocyanate,Sigma) in 1×PBS with 0.2% Triton-X100 for 15 minutes for nuclei andactin cytoskeleton, respectively. Samples were visualized on a LeicaDMRX upright microscope. Prior to visualizing in the SEM, samples werefixed with 2.5% glutaraldehyde in PBS for 1 h at 4° C. washed threetimes with 1×PBS, and dehydrated with a gradient of ethanol at 15%, 30%,50%, 75%, 85%, 90%, 95% and 100%. Dehydrated samples were dried using acritical point dryer, mounted onto stubs with carbon tape and sputtercoated with platinum and palladium for 30 sec. Samples were visualizedin the SEM chamber at an accelerating voltage of 10 kV and spot size 3.

The results of the experiments are now described.

Variation of Fiber Morphology with Solution Concentration

It was observed that SPI/PEO blend fibers as well as zein fibersfollowed the general trend of increasing in fiber width or diameter withincreasing protein concentration. Surprisingly, lower content of PEO didnot correlate to smaller fiber diameters, although the same volume ofsolution yielded less amount of electrospun fibers and the fibers wereless uniform than those from solutions with higher amounts of PEO (SeeFIG. 1 and Table 1). The results presented in Table 1 are summarized inFIG. 2 and FIG. 3. Specifically, FIG. 2 demonstrates the variation offiber diameter with SPI and PEO content; FIG. 3 demonstrates thevariation of fiber diameter with zein concentration.

TABLE 1 Fiber width corresponding to protein concentration % SPI (0.05%PEO) Width (nm) (n = 100) 5 702 ± 201 6 738 ± 195 7 852 ± 373 8 1514 ±447  % zein Diameter (nm) (n = 100) 35 159 ± 18 40 236 ± 21 45 344 ± 27Variation of Fiber Diameters with Hydration

Fibers swelled to more than double their dry diameters after 2 hour ofhydration in the case of SPI/PEO blend fibers. It was observed thatswelling decrease after 24 h. It was observed that 6% SPI, 0.05% PEOfiber widths varied with hydration for 2 hour and 24 hour in DMEM, PBSand H₂O (See Table 2 and FIG. 4). It was observed that 40% zein fiberwidths varied with hydration for 2 hour and 24 hour in DMEM, PBS and H₂O(Table 3).

TABLE 2 6% SPI, 0.05% PEO fiber diameter after hydration DMEM PBS H₂ODry 2 h 24 h 2 h 24 h 2 h 24 h 738 ± 195 1639 ± 291 1555 ± 236 1687 ±456 1549 ± 254 1644 ± 363 n/a

TABLE 3 Zein fiber diameter after hydration DMEM PBS H₂O Dry 2 h 24 h 2h 24 h 2 h 24 h 236 ± 21 342 ± 77Variation of Tensile Properties with Solution Concentration

SPI/PEO blend and zein scaffolds had similar mechanical properties andbreaking mechanisms. Thinner dry specimens tended to break in a clean,brittle manner, while thicker specimens broke gradually as measured bythe layers of fibers shearing apart (data not shown). Minimal differencewas observed between mechanical properties of scaffolds with variationin the protein contents investigated. Hydrated samples had a Young'smodulus of more than an order of magnitude lower than dry samples, witha correspondingly higher elasticity as determined by the higher strainat break. Some specimens did not break at 200% strain.

TABLE 4 Young's Modulus Ultimate Tensile (MPa) Strength (MPa) Strain atBreak (%) dry hydrated dry hydrated Dry hydrated SPI (%) 5  26.47 ±15.19 0.19 ± 0.19 0.92 ± 0.38 0.08 ± 0.04 10.14 ± 2.33  87.74 ± 18.03 626.60 ± 7.58 0.17 0.47 ± 0.14 0.079 4.44 ± 0.35 42.78 7 19.63 ± 4.430.19 ± 0.12 0.40 ± 0.11 0.12 ± 0.08 6.48 ± 1.01 67.51 ± 23.44 8  33.28 ±12.12 0.14 0.75 ± 0.29 0.12 ± 0.01 5.22 ± 0.90 85.83 ± 5.11  Zein (%)35  21.07 ± 6.21 0.75 ± 0.90 0.94 ± 0.39 0.39 ± 0.12 n/a 40  11.63 ±7.08 0.96 ± 0.27 0.51 ± 0.23 0.098 ± 0.015 12.14 ± 5.08  63.48 ± 11.4745  21.59 ± 6.86 0.043 ± 0.012 0.40 ± 0.13 0.066 ± 0.025SPI/PEO and Zein Scaffolds Support Cellular Growth and Proliferation

The next set of experiments were designed to assess whether theengineered scaffolds would support cell growth. Cells were seeded ontoeach respective scaffold (SPI/PEO and zein scaffold). Cells were alsoseeded onto scaffolds electrospun from 20% PLGA and 8% gelatin assynthetic and natural control materials, respectively. It was observedthat human dermal fibroblasts grew able to grow on the engineeredscaffolds (FIG. 5: a) 5% SPI, 0.05% PEO; b) 6% SPI, 0.05% PEO; c) 7%SPI, 0.05% PEO; d) 8% SPI, 0.05% PEO; e) 8% gelatin; f) 20% PLGA.)

Electrospun Scaffolds from Soy Protein Isolate (SPI)/Polyethylene Oxide(PEO) Blend and Zein for Skin Wound Healing

Soy protein has long been exploited for use as industrial replacementsfor more expensive and less eco-friendly materials in textiles,plastics, adhesives and food applications. It has proven to be extremelyversatile in that it can be formulated into films, powders, coatings,solids, gels or fibers depending on the property it is intended toprovide or enhance. From a biological standpoint, soy protein was chosenas a possible biomaterial due to the bioactivity of individual peptidesand isoflavones that may be metabolized by the body upon resorption ofthe scaffold. Despite its versatility, however, the use of soy proteinas a biomaterial has seen slow growth due to processing limitations andpoor mechanical properties. The results presented herein the successfulgeneration of a biomaterial derived from soy bean that can support thegrowth of cells. While soy protein isolate dissolved in bases and1,1,1,3,3,3-hexafluoro-2-propanol (HFP), the solution did not yieldfibers when electrospun, but only electrospraying behavior. Hence, asmall amount of high molecular weight (1,000,000) synthetic polymer,poly(ethylene oxide) (PEO) was added in order to increase the chainentanglements, which resulted in yields of fibers.

Corn zein is an abundant byproduct of the bio-ethanol industry. Theresults presented herein demonstrate the successful generation of abiomaterial derived from Corn zein which was able to support growth ofcells. Electrospinning of zein was optimized using glacial acetic acidas solvent to produce uniform, tubular nanofibers.

The results presented herein shown that SPI, with the addition of asmall amount of PEO, can be formed into stable, hydrolysis-resistantsubmicron fibers that support cellular attachment, growth andproliferation. In addition, zein can be electrospun into stablenanofibers without the need for synthetic additives or crosslinking. Nosignificant difference was observed in the mechanical properties andcellular behavior between SPI/PEO and zein nanofiber scaffolds.

Both SPI/PEO blend fibers as well as zein fibers remained stable inaqueous environments without external crosslinking. The resultspresented herein demonstrate that proteinaceous products from naturalproducts otherwise referred to as “green” proteins provide a platformfor directed wound healing by the induction of native skin fibroblastsand keratinocytes to populate the scaffolds and secrete appropriatematrix components and balanced signals. Although cell culture mediumcontains proteases that may degrade soy proteins and zein over time, theobservation of a more rapid degradation of the scaffolds in mediumcompared to other aqueous solutions such as PBS and water over 8 daysdid not occur. Without wishing to be bound by any particular theory, itis believed that this observation was due to the protective orplasticizing effect of the serum in the medium.

The results presented herein demonstrate that products derived fromalimentary plants, such as soybean and corn amongst other things,provide a platform for skin regeneration with the advantages ofremaining stable without further crosslinking. The results presentedherein describes a novel class of biomaterials.

The disclosures of each and every patent, patent application, andpublication cited herein are hereby incorporated herein by reference intheir entirety.

While this invention has been disclosed with reference to specificembodiments, it is apparent that other embodiments and variations ofthis invention may be devised by others skilled in the art withoutdeparting from the true spirit and scope of the invention. The appendedclaims are intended to be construed to include all such embodiments andequivalent variations.

What is claimed:
 1. An electroprocessed composition comprising: asynthetic polymer and soy protein isolate.
 2. The composition of claim1, wherein said electroprocessed composition is electrospun.
 3. Thecomposition of claim 1, wherein said composition is suitable for use asa wound dressing or skin graft.
 4. The composition of claim 1, whereinsaid composition is capable of supporting cell growth.
 5. Thecomposition of claim 1, wherein said composition is capable ofsupporting the maintenance of a differentiation state of a cell.
 6. Thecomposition of claim 1, further comprising a cell.
 7. The composition ofclaim 6, wherein said cell is genetically modified.
 8. The compositionof claim 1 further comprising a material selected from the groupconsisting of flbronectin, laminin, collagen, glycoprotein,thrombospondin, elastin, fibrillin, mucopolysaccharide, glycolipid,heparin sulfate, chondroitin sulfate, keratin sulfate,glycosaminoglycan, hyaluronic acid, proteoglycan, vitronectin,poly-D-lysine, polysaccharide, and any combination thereof.
 9. Thecomposition of claim 1 further comprising an adhesive.
 10. Thecomposition of claim 1, wherein said synthetic material polymer isselected from the group consisting of poly (epsilon-caprolactone) (PCL),poly (lactic acid) (PLA), poly (glycolic acid) (PGA), copolymers poly(lactide-co-glycolide) (PLGA), polyaniline, poly(ethylene oxide) (PEO),and any combination thereof.
 11. A method of making a compositioncomprising a synthetic polymer and soy protein isolate the methodcomprising: obtaining soy protein isolate and dissolving the soy proteinisolate in a solvent to produce a soy protein solution; combining thesoy protein solution with a synthetic polymer; and subjecting the soyprotein solution to electroprocessing to produced electroprocessedfibers.
 12. The method of claim 11, wherein said electroprocessing iselectrospinning, and wherein said electroprocessed fibers areelectrospun fibers.
 13. The method of claim 11, wherein said soy proteinisolate is blended with the synthetic polymer poly(ethylene oxide)(PEO).
 14. The method of claim 11, wherein said synthetic polymer isselected from the group consisting of poly (epsilon-caprolactone) (PCL),poly (lactic acid) (PLA), poly (glycolic acid) (PGA), copolymers poly(lactide-co-glycolide) (PLGA), polyaniline, poly(ethylene oxide) (PEO),and any combination thereof.
 15. The method of claim 11, wherein saidsolvent is selected from the group consisting of an organic solvent, anacid, a base, an alcohol, and any combination thereof.
 16. The method ofclaim 11, wherein said solvent is selected from the group consisting of1,1,1,3,3,3-hexafluoro-2-propanol (HFP) and glacial acetic acid.
 17. Amethod of culturing a cell with an electroprocessed scaffold comprisingcontacting an electroprocessed synthetic polymer and soy protein isolatescaffold with said cells in the presence of a culture medium.
 18. Themethod of claim 17, wherein said culturing a cell with said scaffoldproduces a target tissue substitute.
 19. The method of claim 17, whereinsaid cell is selected from the group consisting of stem cells, musclecells, endothelial cells, nerve cells, bone cells, heart cells,epithelial cells, fibroblasts, and mixtures thereof.
 20. A method ofdelivering an agent to a mammal, said method comprising administering anelectroprocessed synthetic polymer and soy protein isolate scaffold to amammal wherein said scaffold further comprises an agent.
 21. The methodof claim 20, wherein said agent is a cell.
 22. The method of claim 20,wherein said agent is selected from the group consisting of anextracellular matrix component, a growth factor, a differentiationfactor, and combinations thereof.
 23. The method of claim 20, whereinsaid agent is selected from the group consisting of a chemical agent, apharmaceutical, a peptide, a nucleic acid, and any combination thereof.